Testing Status of Agents at NTP
Executive Summary TCAB
1. Human Data
No data were found on the chemical disposition of
TCAB or TCAOB in humans.
2. Animal Data
- oral, rat (TCAB/TCAOB)
-
° An unspecified number of male Sprague-Dawley outbred albino
rats were administered 10 milligrams of carbon-14 labelled TCAB
or TCAOB (249.3 µCi/mmol, 10 mg/ml corn oil) by stomach intubation.
Urine and fecal samples were collected every 24 hours. Rats were
sacrificed on day 5. Based on liquid scintillation spectrometer
counts, TCAB was found to clear from the body more rapidly than
TCAOB in 24-hour urine and feces samples (66% and 37% clearance,
respectively). Based on this rapid elimination phase, the half-lives
for the elimination of TCAB and TCAOB were determined to be 18
and 34 hours, respectively. The terminal phase half-lives for
both compounds were determined to be greater than 20 days. The
authors suggest that this data demonstrate the potential for these
compounds to bioaccumulate under chronic exposure conditions.
For both compounds, the feces was the major route of excretion.
After 48 hours, TCAB and TCAOB levels (% of radioactive dose administered)
in the feces were 55.1±4.9% (TCAB) and 50.0±14.3% (TCAOB).
Levels of TCAB and TCAOB in the urine were 27.1±3.8% (TCAB)
and 20.1±1.8% (TCAOB) after 48 hours. For both compounds
the highest levels of radioactivity were found in the fat (2.70±1.17%
for TCAB and 4.97±2.83% for TCAOB). High concentrations of
radioactive TCAB and TCAOB were also found in the pancreas, lymph
nodes, kidney, liver, and bladder. The lowest concentrations of
radioactivity were found in the brain (0.12±0.01% for TCAB
and 0.09±0.02% for TCAOB) [Burant and Hsia, 1984].
- intraperitoneal, rat microsomes,TCAB
- ° Rat liver microsomes were prepared from male Sprague-Dawley rats given an intraperitoneal injection of TCAB at a concentration of 25 mg/kg/day mixed in corn oil for five days. Control rats received 5 ml/kg/day corn oil only. A mixture containing carbon-14 labelled TCAB (112-138 µg, 128 µCi/mmol), bovine serum albumin (2 mg), and dimethyl sulfoxide (DMSO) (1% v/v) was added to an NADPH-generating system. Samples designated as controls used heat-deactivated microsomes. Nearly all the radioactivity added to the incubation system was recovered. The rate of TCAB metabolism was determined to be 381 ± 59 pmol/min/mg microsomal protein. The major metabolite detected was a TCAB phenol. Other metabolites detected included N-hydroxy-3,3',4,4'-tetrachlorohydrazobenzene and 3,3'4,4'-tetrachlorohydrazo-benzene. In order to gain insight into the underlying biochemistry of the formation of the TCAB phenol, and the concurrent binding with the macromolecule pellet, the monooxygenase activity in the incubation system was modulated. Monooxygenase inhibitors, carbon monoxide and 2-diethylaminoethyl 2, 2-diphenyl-valerate hydrochloride were added to the system and the system was deprived of NADPH. Under these conditions, a significant reduction in TCAB phenol formation and covalent binding was observed [Hsia and Kreamer, 1981].
B. Acute
1. Human Data
No data were found on the acute toxicity of TCAB
or TCAOB in humans.
2. Animal Data
- oral, dermal,inhalation, rat(TCAB)
-
°
An acute oral rat LD50 of > 5000 mg/kg (0/10 deaths), a skin
approximate lethal dose (ALD) in rabbits of > 1000 mg/kg (0/6
deaths), and an inhalation (4-hour) acute lethal concentration
(ALC) in rats of 0.92 mg/l have been reported for TCAB [Taylor
and Lloyd, 1982]. No other data were provided.
E.I. du Pont de Nemours & Company (Haskell Laboratories) conducted the following acute toxicity test on laboratory animals:
- dermal, rabbit(TCAB)
-
°
Pairs of rabbits of unspecified sex and strain received an application
of either TCAB, 3,4-dichloroaniline (DCA) containing 5% tar, DCA
containing 8% TCAB, chloroform (negative control), dimethyl sulfoxide
(negative control) in 5% chloroform solutions, or 0.1 milliliters
of a Dow positive control on the left external ear canal. Right
ears of each rabbit were treated with 0.1 milliliters of chloroform
only. Two days after the test application, rabbits were sacrificed.
Upon pathological analysis, rabbits that received TCAB, DCA containing
5% tar, and DCA containing 8% TCAB had marked epidermal hyperplasia,
sebaceous gland hyperplasia, squamous metaplasia, and dilation
of hair follicles (with an increase in keratin material, fibrosis
of dermis, and some inflammatory infiltrate). Thickening of the
skin and possible systemic effects were also noted. Chloroform
produced similar but less pronounced changes. The TCAB-containing
compounds were determined to be acnegenic [E.I. du Pont de Nemours
& Company, Inc., 1982b].
- dermal, rabbit(TCAB)
- ° Table 3 summarizes results of rabbit ear bioassays following the application of TCAB at various concentrations [Hill et al., 1981].
| Compound Tested | Formulation for Ear Test mg/ml (MIBK)a | TCAB Content of Sample (µg/g) | Total dose TCAB Applied (µg) | Microscopic Evaluation of Rabbit Ear Testb |
|---|---|---|---|---|
| TCAB | 0.001 | -- | 0.5 | -,+ |
| TCAB | 0.01 | -- | 5 | +,++ |
| TCAB | 0.02 | -- | 8 | +,++ |
| TCAB | 0.04 | -- | 16 | ++,++ |
| TCAB | 0.08 | -- | 32 | ++,++ |
NOTES:
- = no hyperkeratosis
+ = mild hyperkeratosis
++ = moderate hyperkeratosis
a) MIBK = methyl isobutylketone
b) Two rabbits per test were evaluated. If both "treated" ears reacted the same, only one rating was given; if they reacted differently, both ratings were given.
___________
Reference: Hill et al., 1981
C. Prechronic
1. Epidemiological Evidence/Case Reports
A number of cases of chloracne have been reported
following occupational exposure to herbicides. In these cases,
the observed chloracne was attributed to TCAB and/or TCAOB. In
general, chloracne is characterized by comedone formation, straw-colored
cysts, and inflammatory papules. The most sensitive area of the
skin is around the eyes and ears. Chloracne may be associated
with systemic toxicity. Direct skin contact is expected to be
the primary route of exposure; however, inhalation and ingestion
are speculative routes.
- occupational,human (TCAOB/methazole)
- ° One of the first reports of TCAOB-induced chloracne involved
41 production workers (average age 29) in a chemical manufacturing
plant. Workers developed bumps within 1-2 months after they began
manufacturing 2-(3,4-dichlorophenyl)-4-methyl-1,2,4-oxadiazolidine-3,5-dione
(methazole) which contains unspecified concentrations of TCAOB
as a contaminant. Ninety percent of the workers developed chloracne.
Of the 41 workers who developed chloracne lesions, 38 had lesions
on the face, 33 on the neck, 31 on the arms, 31 on the legs, 27
on the trunk, and 15 on the genitals. No significant differences
were found in serum glutamic oxaloacetic transaminase or porphyrin
levels. Four family members of four workers who had never been
inside the plant developed chloracne. This may have resulted
from direct contact with contaminated clothing or tools [Taylor
et al., 1977].
Seven to eight years later, a long-term follow-up study was conducted on 5 of the workers and 2 children who had developed chloracne. Three of the workers still had evidence of chloracne. Four of the five workers were sensitive to sunlight. The 2 children had mild scarring, and one of the children (a 15-year-old girl) had acne vulgaris [Taylor and Lloyd, 1982].
- occupational,human (TCAB/methomyl,propanil)
-
° In a methomyl (1-(lamda-methylthio)ethylideneamino methyl
carba-mate)/propanil (pesticides contaminated with TCAB) pesticide
manufacturing plant, 102 of 111 employees (plant workers and office
workers) participated in a medical survey. Ninety six percent
of the workers were male (88% white). The average employee age
and length of employment were 28.7 years and 24 months, respectively.
Among the participating employees, 6.9% had chronic health problems.
Workers involved in the production of propanil had the highest
rate of chloracne with an average of 1.39 symptoms per worker.
Symptoms reported among the 28 production workers included acne
(78%), rash/skin irritation (46%), eye irritation (25%), and cyanosis
(21%). In 101 workers, symptoms included acne (39%), rash/irritation
(34%), eye irritation (26%), and cyanosis (68%). Other symptoms
observed included small pupils, nausea/vomiting, blurred vision,
muscle weakness, coughing, headache, fatigue, confusion, increased
salivation, and asthma. Seventeen (61%) of the 28 production workers
were hospitalized due to chloracne. No significant hematological
abnormalities were noted. In addition, cholinesterase activity
was not affected by propanil exposure [Morse and Baker, 1979].
- occupational,human (TCAB)/2,4,5-T
- °
In an epidemiological case control study, 54 pesticide applicators
who sprayed 2,4,5-trichlorophenoxy acetic acid (2,4,5-T) (a herbicide
contaminated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or
TCAB for 63 weeks were compared with 54 workers who did not spray
2,4,5-T. No significant differences in liver function, porphyrin
excretion, or prevalence of acne were found in the exposed compared
to the non-exposed group [Houdt et al., 1983].
- occupational,human (TCAB/TCAOB)
- ° Table 4 presents data on the occurrence of chloracne outbreaks from 3,4-dichloroaniline derived herbicides containing TCAB and/or TCAOB [Taylor and Lloyd, 1982].
| Location | Year | Herbicide | Source | Number of Workers (%) | Probable Chloracnegenic Chemical |
|---|---|---|---|---|---|
| Illinois | 1960s and/or 1970s | Methazole | Chemical Plant | <10 | TCAOB |
| England | 1970s | Methazole | Chemical Plant | U | TCAOB |
| Ohio*1 | 1972-73 | Methazole | Chemical Plant | 41 (90%) | TCAOB |
| Michigan | 1970s | Methazole | Chemical Plant | U | TCAOB |
| New Jersey | 1960s and/or 1970s | Propanil | Chemical Plant | U | TCAB |
| Southern U.S.A. | 1960s and/or 1970s | Propanil | Railroad cars delivering herbicide | U | TCAB |
| Arkansas**1 | 1974-77 | Propanil | Chemical Plant | 17 (61%) | TCAB |
| Europe | 1970s | U | Chemical Palnt | U | TCAB |
U--unknown; *Taylor and co-workers, 1974; **Morse and co-workers, 1977, 1979.
1--Previously described.
___________
Reference: Taylor and Lloyd, 1982
2. Animal Data
- intraperitone al,rat, mouse(TCAB/TCAOB)
-
°
Three groups of male Sprague Dawley rats (n=4) were injected intraperitoneally
with 25 mg/kg TCAB 4 times per day, or with TCAOB 2 times per
day in corn oil on days 1 and 5. Animals that received TCAB and
TCAOB were sacrificed on days 5 and 10, respectively. Control
rats received 2.5 ml/kg corn oil only. In addition, a group of
female ICR outbred Swiss albino mice of unspecified number were
injected intraperitoneally with 20 mg/kg TCAB, 5 times per day
following lactation. On day 6, the mice were sacrificed.
In mice and rats, both compounds caused histopathological changes of the liver, including hypertrophy and hyperplasia (greater than 50% compared to controls). Liver cells of the TCAB and TCAOB treated rats had granular cytoplasm containing vacuoles; some hepatocytes contained mitotic figures (P < 0.001). In addition, TCAB and TCAOB treatment induced proliferation of the smooth endoplasmic reticulum of rat liver cells. Mice livers treated with TCAB developed mitotic figures in which chromosomes appeared in a tripolar arrangement [Schrankel et al., 1980].
- dermal,mouse, rabbit(TCAOB)
-
°
The chloracnegenic potential of TCAOB was investigated in five
strains of mice including the hairless rhino (develops spontaneous
follicular hyperkeratosis), rhino+ (does not develop spontaneous
follicular hyperkeratosis), DBA/2J (an aryl hydrocarbon hydroxylase
(AHH) enzyme induction insensitive strain), and C57BL/6 (an AHH
enzyme induction sensitive strain). Male New Zealand white rabbits
served as the positive control as this strain appears to be the
most sensitive and reliable animal model for chloracne. Three
studies were conducted using 3-5 mice of each strain per dose.
In studies 1, 2, and 3, (see below) mice received test doses 5
days per week for 3 to 9 weeks (depending on when chloracne developed)
on the hair-clipped dorsa. TCAB was applied to the inner surface
of the right ear and control doses were applied to the left ear
in all animals.
In study 1, male mice from each strain were tested with 0.001% TCAOB in acetone 5 days per week for 9 weeks. Termination of the experiment was based on mortality. Three positive control rabbits were treated with 0.001% TCAB as follows: 1 per week for 17.5 weeks; 3 times per week for 17.5 weeks; or 5 times per week for 6 weeks.
In study 2, female rhino and hairless mice received daily applications of 0.001%, 0.01%, and 0.1% TCAOB five days per week until fatalities occurred. Two positive control male rabbits received 0.001% TCAOB daily and 100 microliters of acetone.
In study 3, male hairless mice and rhino mice received doses of 0.01 and 0.1% TCAOB. Rhino mice received 18 treatments (3.5 weeks) at both dose levels and hairless mice received 12 treatments of 0.1% TCAOB or 18 treatments of 0.01% TCAOB.
Based on gross and histologic examination of the skin, no abnormalities occurred in treated mice from study 1. However, signs of chloracne, including hyperplasia and hypertrophy of the right ear, were seen in rabbits within 2.5 weeks. A dose-dependent follicular hyperkeratosis and epithelial hyperplasia (characteristic of a chloracnegenic response) were seen in mice in studies 2 and 3. Mice receiving 0.01% and 0.1% TCAOB experienced more severe effects including erythema, skin-fold thickening, discoloration, and weight loss. In studies 2 and 3, eyes of the rhino mice treated with TCAOB were swollen shut with keratinaceous ocular discharge. Rabbits also developed chloracne. One male hairless mouse in the 0.1% TCAOB dose group and one rhino mouse in the 0.01% TCAOB dose group died at 3 and 4 weeks, respectively. Rhino mice in the 0.1% TCAOB dose group developed necrotic foci of the liver [Horton and Yeary, 1985].
- dermal, rabbit(TCAB)
-
°
Albino rabbits of unspecified sex and number received applications
of 0.0001%, 0.001%, or 0.01% TCAB dissolved in acetone solutions
to the ear five days per week for 4 weeks. Fifty percent Halowax
1041 in mineral oil and acetone served as positive and negative
controls, respectively. Comedone formation was observed in all
TCAB-treated rabbits. Comedone response in the 0.0001% TCAB-dosed
rabbits closely resembled Halowax-treated comedone formation.
No comedones were seen in the acetone treated rabbits [Taylor
et al., 1977].
E.I. du Pont de Nemours & Company, Inc., (Haskell Laboratories), conducted the following prechronic skin absorption studies in albino rabbits of unspecified strain:
- dermal, rabbit(TCAB)
-
°
In the first part of the study, groups of male rabbits (n=10)
received an application of 20 mg/kg TCAB in a 10% solution of
acetone or 5 milliliters of acetone as a control. Control and
test materials were applied by wrapping to the clipped shoulders
and backs of the rabbits for 6 hours per day for 10 days. After
6-8 days of administration, 3/10 deaths had occurred in the test
group. Animals appeared lethargic and had cold extremities prior
to death. The surviving animals were necropsied on day 15. Blood
was taken from the marginal ear vein of each rabbit prior to the
application of test material and after the fifth and tenth applications.
All rabbits developed moderate to severe skin irritation with
fissuring and thick, crusty, and necrotic skin. The authors concluded
that this effect was due to acetone. Clinical observations included
decreased hematocrit values, hemoglobin levels, and erythrocyte
counts compared to controls on days 5 and 8. Test group rabbits
had an increased level of glutamic pyruvic transaminase activity.
All animals had an increased methemoglobin level by day 10. From
gross pathologic examination it was determined that death occurred
due to liver toxicity. The livers appeared swollen and soft with
fatty infiltration. The test group had slightly enlarged livers.
° In the second part of the study, 40 male albino rabbits were divided into groups in order to establish a no-effect level for "Still Bottom Tars." Groups 1 and 2 were divided into 2 dose groups. Group 1a (n=15) (control) received 3.5 milliliters of acetone for 10 days (wrapped). Group 1b (n=10) received 0.35 milliliters of acetone for 20 days (not wrapped). Group 2 (n=10) received 0.35 milliliters TCAB in 0.1% acetone solution (unwrapped) for 20 days. The day after the 10th treatment, five animals from each group were sacrificed. The remaining rabbits were sacrificed on day 14 or 30. Blood was taken from the marginal ear vein before the test began, after the 10th treatment, after the 30th treatment (group 1 and 2), and 14 days after the last treatment. By day 6, severe skin irritation had developed in all test groups with characteristics similar to those described for the first part of the study. [E.I. du Pont de Nemours & Company, Inc., 1982f].
- dermal, rabbit(TCAB)
-
°
Eight groups of rabbits (n=2) of unspecified strain or sex received
approximately 0.1 milliliters of TCAB at a concentration of 0.002%,
0.02%, 0.2%, or 2.0% in 2,3-dichloroaniline (2,3-DCA); a solution
of 3,4-dichloroaniline (DCA) 50% by weight in chloroform; a solution
of DCA 50% by weight in 2,3-DCA; pure 2,3-DCA (99.25%); or chloroform.
The solution of DCA 50% by weight in chloroform and 2,3-DCA both
contained 23 ppm TCAB. TCAB in 2,3-DCA served as the positive
control. All solutions were applied to the distal inner right
ear of each rabbit, 5 days per week for 4 weeks. One animal per
group was sacrificed after a 2-day rest period.
TCAB (concentrations ranging from 0.002%-2.0% TCAB in 2,3-DCA) and DCA were acnegenic. DCA 50% by weight in chloroform and DCA 50% by weight in 2,3-DCA produced only mild acnegenic effects accompanied by pore enlargement. Gross examination of the skin revealed erythema, sloughing, and ear thickening. Pathological findings included hair follicles filled with yellow plugs and keratinous material on the skin [E.I. du Pont de Nemours & Company, Inc, 1982a].
- dermal, rabbit(TCAB)
-
°
Eight groups of rabbits of unspecified strain and sex received
applications of 3,4-dichloroaniline (DCA) "Incident Tars"
at concentrations of 0.01%, 0.1%, 1.0%, and 10.0% in chloroform,
and TCAB at concentrations of 0.002%, 0.02%, 0.2%, and 2.0% in
chloroform on the distal half of the inner left ear five days
per week for 4 weeks. The lowest concentrations of both compounds
were continued for 2 additional weeks. Both compounds were found
to induce strong dose-dependent acnegenic responses including
skin sloughing and thickening and plug formation of the skin.
However, "Incident Tars" were 1/5 as active as pure
TCAB. The NOEL for incident tars and TCAB was determined to approach
0.01% and 0.002%, respectively [E.I. du Pont de Nemours &
Company, Inc., 1982d].
- dermal, rabbit(TCAB)
- ° Five groups of rabbits (n=2) of unspecified strain and sex received an application of approximately 0.1 milliliters of 3,4-dichloronitrobenzene, 3,4-dichloroaniline (DCA), DCA in 10% chloroform solution, a 10% solution of TCAB in chloroform, a positive control Halowax 50% suspension in chloroform, or a negative (chloroform) control to the left ear, 5 days per week for 4 weeks. DCA was found to be acnegenic, producing skin sloughing, thickening and plug formation. TCAB was found to be a strong acnegen, causing sloughing, crusty skin, erythema, thickening, plug formation, and hair loss. Symptoms worsened with time. Slight sloughing and erythema was observed in the negative control group [E.I. du Pont de Nemours & Company, Inc., 1982c].
1. Human Data
No data were found on the chronic/carcinogenic effects
of TCAB or TCAOB in humans.
2. Animal Data
- oral, rat(TCAB/TCAOB)
-
°
Groups of 10 male Sprague Dawley rats were fed a diet containing
100 ppm TCAB or TCAOB in corn oil (control) for 120 days. On the
last day of the experiment, animals were anesthetized and blood
was collected. Food consumption and body weights were measured
2 and 3 times weekly, respectively. TCAB and TCAOB intake over
the entire study was 25.2 ± 2.4 milligrams and 24.0 ±
2.3 milligrams, respectively.
A significant decrease in body weight was seen in the TCAB (9.4%) and TCAOB (16.9%) treated rats compared to controls. Hematocrit values and hemoglobin levels decreased in test groups. This decrease was more significant in TCAOB-treated rats (P < 0.001) than TCAB treated rats (P < 0.05). The white blood cell count was insignificantly decreased in both treated groups. The red cell count was significantly decreased in the TCAOB group (P < 0.001) and insignificantly decreased in the TCAB treated group. Liver, spleen, and testicular weights increased significantly (P < 0.05, P < 0.05, and P < 0.005, respectively) in TCAOB treated rats compared to controls. These organ weights were insignificantly increased in TCAB treated rats. Biochemical measurements conducted in this study are reported in section VG.3 [Hsia et al., 1980].
E. Reproductive Effects and Teratogenicity
1. Human Data
No data were found on the reproductive or teratogenic
effects of TCAB or TCAOB in humans.
2. Animal Data
- oral, mice(TCAOB)
-
°
Bleavins et al. (1985a) conducted a reproductive/immunocompetence
study in order to evaluate the effects of in utero and
early postnatal exposure to TCAOB on pup survival and immune function,
and on the reproductive efficiency of their dams. The doses were
selected so that no overt indications of toxicity were likely
to be observed in the adult females. These doses were based on
the results of a previous immunotoxicity study of TCAOB in young
female mice that was conducted by Bleavins et al. (1985b).
Four groups of 13 adult virgin female Swiss-Webster mice were
administered for 14 days, 0 (control), 0.1 ppm, 1 ppm, or 10 ppm
TCAOB dissolved in corn oil and mixed in powdered feed. The females
were mated with untreated males on day 14, and pregnant females
were continued on the test diet until delivery. On day 28 postpartum,
females and pups were sacrificed in order to measure immune function
in the pups, and to obtain organ weights in the mothers and offspring.
Immune parameters including thymus weight and plaque forming cells
(PFCs) per leukocyte and per spleen were measured. Immunocompetence
among the offspring was determined by injecting pups with sheep
red blood cells (SRBCs) on day 23 post partum and measuring immune
parameters.
Adverse effects in the treated groups were compared to controls. The only maternal toxic effect observed was a significant (P < 0.05) decrease in the thymus weight in the 10 ppm TCAOB treated group. A significant (P < 0.01) decrease in the number of pups per female whelping at birth and at weaning was observed in the 10 ppm TCAOB treated mice. A significant (P < 0.01) increase in pup weight at birth compared to controls was observed in the 1 ppm TCAOB treated group. The sum of the individual pup weights (litter mass) was significantly decreased at birth and on days 7, 14, 21, and 28 post-partum (P < 0.01, P < 0.05, P < 0.01, P < 0.01, and P < 0.01, respectively) in the 10 ppm TCAOB treated group. A significant (P < 0.05) decrease in the litter mass was also observed in the 1 ppm TCAOB treated group on day 21.
In the 28-day-old pups used to assess immune function, thymus weights were insignificantly less than controls. However, the thymus weights of the 10 ppm TCAOB treated pups not immunized with SRBCs were significantly (P< 0.01) lower than control pups. In the immunized mice, no significant difference in liver and spleen weights was observed compared to control mice. However, plaque forming cells were significantly decreased (P < 0.01) compared to control mice [Bleavins et al., 1985a].
- intraperitoneal,mice (TCAOB)
-
°
The teratogenicity of TCAOB was studied in Ah-responsive
and non-responsive mice. Three-month-old Ah-responsive
(C57BL and NMRI) and Ah-nonresponsive (AKR/NBom and DBA/2J)
mice were mated. Pregnant females were injected intraperitoneally
with 6, 8, or 16 mg/kg TCAOB dissolved in dioxane on days 10-13
of gestation. The control group received 320 µl/kg dioxane
only. On day 17, animals were sacrificed and their uteri were
examined for number of implantations, dead, resorbed or alive
fetuses.
In addition, pregnant C57BL mice were treated with dioxane or TCAOB at 8 mg/kg on day 12, or cortisone acetate at 2.5 mg/animal on days 11-14 and sacrificed on day 15. The embryos were removed and heads were prepared for electron microscope viewing in order to examine palate cells.
The specific dosing scheme and results from the experiments outlined above are presented in Table 5.
As part of the same study, the effect of TCAOB in the offspring of the above matings (AKR X C57BL; C57BL X AKR; NMRI X DBA) was compared to the effect of this compound on inbred parental strains. Backcrosses between the F1 generation of NMRF and DBW with inbred NMRI was also tested. The specific treatment regimen and experimental results are described in Table 6.
of Teratology Study
| Strain | Day Treated (3 p.m.) | Dosage | Dams with malformed fetuses %, (no. of affectedb/treated) | No. of implan- tations | Resorption + dead fetuses % (early/late)c | Cleft Palate %d | Hydro- nephrosis % | Hydrops % |
|---|---|---|---|---|---|---|---|---|
| C57BL | 10 | TCAOB 6 mg/kg | 100 (11/11) | 77 | 31.2 (15/9) | 33.6 | 63 | 0 |
| 10 | Dioxanea | 0 (0/7) | 46 | 19.6 (9/0) | 0 | 0 | 0 | |
| 11 | TCAOB 6mg/kg | 100 (10/10) | 77 | 28.6 (15/7) | 64.7 | 79.3 | 5.1 | |
| 11 | Dioxanea | 28.6 (2/7) | 52 | 15.4 (7/1) | 4.5 | 0 | 0 | |
| 12 | TCAOB 6mg/kg | 100 (11/11) | 80 | 21.3 (9/8) | 56.6 | 37.3 | 8.1 | |
| 12 | TCAOB 16mg/kg | 66.7 (6/9) | 50 | 60 (29/1) | 95 | __ | 2 | |
| 12 | Dioxanea | 40 (2/5) | 35 | 14.3 (5/0) | 0 | 12.5 | 0 | |
| 13 | TCAOB 6mg/kg | 90.9 (10/11) | 82 | 19.5 (8/8) | 23 | 26.9 | 7.9 | |
| 13 | Dioxanea | 0 (0/5) | 33 | 18.2 (6/0) | 0 | 0 | 0 | |
| DBA | 10 | TCAOB 8mg/kg | 28.6 (2/7) | 54 | 5.6 (2/1) | 1.9 | 2 | 0 |
| 11 | TCAOB 8mg/kg | 28.6 (2/7) | 50 | 20 (10/0) | 5 | 0 | 0 | |
| 12 | TCAOB 8mg/kg | 14.3 (1/7) | 48 | 8.3 (4/0) | 2.3 | 0 | 0 | |
| 12 | TCAOB 16mg/kg | 11.1 (1/9) | 52 | 38.4 (14/6) | 3.1 | __ | 0 | |
| AKR | 10 | TCAOB 8mg/kg | 12.5 (1/8) | 38 | 10.5 (4/0) | 0 | 2.9 | 0 |
| 11 | TCAOB 8mg/kg | 25 (2/8) | 34 | 14.7 (5/0) | 0 | 10.3 | 0 | |
| 12 | TCAOB | 28.6 (2/7) | 40 | 7.5 (3/0) | 2.7 | 2.7 | 0 |
a Dioxane used as solvent for the TCAOB
b Malformations only
c Dead Fetuses less than about 6 mm of length have been assigned to the group of early dead
d Based on fetuses being alive or dead in late stage and possible to investigate
___ Not investigated
__________
Reference: Hassoun et al., 1984
| No. of live fetuses | Cleft palate % of live fetusesa |
|||||||
|---|---|---|---|---|---|---|---|---|
| Strain | Dose mg/kg | No. of dams | No. of implan- tations | Resorptions + dead fetuses % | Non- pigm. | Pigm. | Non- pigm. | Pigm. |
| 1) C57BL | 6 | 11 | 80 | 21.3 | __ | 63 | __ | 50.8 |
| 2) AKR | 8 | 7 | 40 | 7.5 | 37 | __ | 2.7 | __ |
| 3) C57BL fx AKR m | 6 | 12 | 84 | 11.9 | __ | 74 | __ | 1.4 |
| 4) C57BL fx C57BL m | 10 | 10 | 73 | 16.4 | __ | 61 | __ | 1.6 |
| 5) AKR fx C57BL m | 6 | 9 | 19 | 10.5 | __ | 17 | __ | 0 |
| 6) (AKR x C57BL) fx AKR m | 10 | 10 | 95 | 4.2 | 47 | 44 | 2.2 | 0 |
| 7) AKR fx (AKR x C57BL) m | 10 | 10 | 55 | 18.2 | 21 | 24 | 4.8 | 8.3 |
| 8) NMRI | 8 | 16 | 147 | 8.8 | 134 | __ | 90.3 | __ |
| 9) DBA | 8 | 7 | 48 | 8.3 | __ | 44 | __ | 2.3 |
| 10)NMRI fx DBA m | 8 | 12 | 102 | 9.8 | __ | 92 | __ | 6.5 |
| 11)NMRI fx (NMRI x DBA) m | 8 | 16 | 148 | 9.5 | 66 | 68 | 48.5b | 51.5b |
| 12)(NMRI x DBA) fx NMRI m | 8 | 17 | 150 | 5.3 | 61 | 80 | 18b | 23.8b |
f =female
m =male
aSee Table 5
bPercent malformations among combined black and white offspring of NMRI fx (NMRI x DBA) m significantly different
from that of (NMRI x DBA) fx NMRI m
P<0.01
__________
Reference: Hossoun et al., 1984
No evidence of maternal toxicity was observed in
either the Ah-responsive or Ah-nonresponsive mice.
C57BL mice treated with 6 mg/kg TCAOB on days 10, 11, 12, or 13
of gestation had an increased percentage of malformed fetuses
(90.9-100%). Malformations observed included cleft palate, hydronophrosis,
and hydrops in several cases (see Table 5). The percentage of
hydrops increased in C57BL mice following treatment with 6 mg/kg
TCAOB on days 11, 12, and 13 of gestation. Treatment of C57BL
mice with 16 mg/kg TCAOB on day 12 of gestation resulted in a
60% occurrence of resorbed and dead fetuses, and a 95% occurrence
of cleft palate. The frequency of the four parameters of fetal
toxicity presented in Table 5 changed with time of administration
in the C57BL dams. Generally, TCAOB treatment resulted in high
frequency of late fetal death. An exception to this was observed
in the group treated with 16 mg/kg TCAOB, where the frequency
of hydrops showed a tendency to increase (not significantly) when
the TCAOB was given late. Treatment of DBA mice with 16 mg/kg
TCAOB caused an increase in resorption and fetal death (38%) compared
to controls; however, no increase in cleft palate was noted. No
evidence of toxicity was seen in the AKR mice after treatment
with 8 mg/kg TCAOB on days 10, 11, or 12 of gestation. The authors
suggested that this increase in sensitivity to TCAOB treatment
by the Ah-responsive mice demonstrates an involvement of
the Ah-locus in cleft palate formation.
The cross breeding study indicated that "the
nonresponsiveness of DBA and AKR mice segregates as a dominant
trait" in the crosses with C57BL and NMRI, respectively.
The authors suggest that the percentage of malformations was significantly
higher among the backcross fetuses where the mother was an inbred
NMRI (father NMRI x DBA) compared to the situation where the mother
was NMRI x DBA (father inbred NMRI). Provided that the sensitivity
is not linked to the sex chromosomes, the authors suggest that
the host maternal factor is involved in the teratogenic mechanism.
NMRI mice had a high incidence of cleft palate formation in the
8.0 mg/kg TCAOB dosed group on day 12 of gestation. The authors
also assert that because approximately 20% of the offspring had
cleft palate after TCAOB treatment, the fetal genotype may be
determined by sensitivity to the teratogenic action of TCAOB.
Examination by scanning electron microscopy of the
palate cells from embryos of pregnant C57BL mice treated on day
14 with TCAOB did not reveal degeneration [Hassoun et al.,
1984].
- intraperitoneal,mice (TCAOB)
-
°
Eight NMRI (Ah-responsive) and DBA2J (Ah-nonresponsive)
3-month-old pregnant mice were sacrificed on day 3 of gestation,
and their uteri were excised. A reciprocal blastocyte transfer
was performed on day 2 of the host gestation. On day 12 of gestation,
mice were injected intraperitoneally with either 2,3,7,8-tetrachlorodibenzo-p-dioxin
(TCDD) or TCAOB dissolved in dioxane at doses of 30 µg/kg
and 8 mg/kg, respectively. On day 16 or 17 of gestation, animals
were sacrificed and their uteri were examined for the number of
implantations and dead, resorbed or live fetuses, and the occurrence
of cleft palate.
None of the DBA fetuses (including those transferred into NMRI uteri) developed cleft palates. Of the NMRI fetuses that remained in the NMRI uteri following treatment with TCDD, 85% (29/34) had cleft palate while 100% (11/11) of the NMRI fetuses in the DBA dams had cleft palate. In the TCAOB treatment group, 90% (56/62) of the NMRI fetuses that remained in the NMRI uteri developed cleft palate and 93% (13/14) developed cleft palate following transfer to a DBA uterus [D'Argy et al., 1984].
- intraperitoneal,mice (TCAOB)
-
°
Three groups of 3-month-old NMRI pregnant mice were injected intraperitoneally
with 3 doses of D,L-alpha-difluoromethyl ornithine (DFMO)
dissolved in saline at concentrations of 100, 200, or 300 mg/kg
at 12-hour intervals, or 3 doses of 5000 µg/kg saline on
days 11 or 12 of gestation. The control group received no treatment
(n=10). A dose of 4 mg/kg TCAOB dissolved in dioxane was injected
into the saline (n=14) and DFMO (n=18) treated mice on days 11
or 12 of gestation. In addition, some DFMO treated animals (n=10)
were injected intraperitoneally with dioxane (300 µl/kg).
Animals were weighed and sacrificed on day 17 of gestation and
their uteri were examined for the number of implantations as well
as dead, reabsorbed, and live fetuses.
Maternal toxicity from TCAOB or DFMO treatment was not observed. The effects of TCAOB treatment on NMRI pregnant mice on day 11 and DFMO on days 11 through 12 of gestation are presented in Table 7. The effects of the treatment of pregnant NMRI mice with TCAOB on day 12 of gestation and DFMO on days 12 through 13 of gestation are presented in Table 8. Administration of DMFO at any dose produced no cleft palates. At a dose of 300 mg/kg, DMFO increased the rate of fetal death compared to that observed in the vehicle control group (P < 0.02). DFMO decreased the frequency of cleft palate when co-administered with TCAOB. TCAOB-induced fetal death was not affected by DFMO administration. The authors concluded that the possible mechanism of cleft palate formation involves TCAOB stimulation of the polysubstrate monooxygenase system and other enzymes of the Ah-locus system, thus keeping epithelial cells alive leading to the formation of cleft palate. DFMO inhibits the activity of this enzyme and thereby decreases cleft palate formation [Hassoun and Arif, 1988].
| Dosage | Dams with Fetuses Having Cleft Palate % and (Number of Affected/Treated) | Mean of Implantation Number/Dam ± S.E.M. and (Total Number) | Percent of Fetuses/Dam Being Resorbed or Dead ± S.E.M. and (Number Early/Number Late)a | Percent of Fetuses/Dam Having Cleft Palate ± S.E.M. and (Number Among Investigated) |
|---|---|---|---|---|
| No Treatment | 0 (0/11) | 6.1 ± 0.73 (67) | 11.9 &3177; 4.3 (7/1) | 0 (0/60) |
| DFMO + dioxane | 0 (0/10) | 8.8 ± 0.56 (88) | 13.6 ± 3.4 (10/0) | 0 (0/78) |
| TCAOB + saline | 42.9 (6/14) | 7.6 ± 0.62 (106) | 33.9 ± 8.1* (25/11) | 37.0 ± 7.4 (30/81) |
| TCAOB + DFMO | 33.3 (6/18) | 8.9 ± 0.43 (160) | 36.3 ± 10.7 (38/20) | 17.2 ± 3.2** (21/122) |
a Resorbed plus dead fetuses (<6 mm of length) have been assigned to the group of early dead, while dead fetuses more than 6 mm of length have been assigned to the group of late dead.
* Significantly different from control (DMFO-plus dioxane-treated), by Student's t-test, P = 0.025.
** Significantly different from control (saline-plus
TCAOB-treated), by Students's t-test, P < 0.01.
| Dosage | Dams with Fetuses Having Cleft Palate % and (Number of Affected/Treated) | Mean of
Implan- tation Number/ Dam ± S.E.M. and (Total Number) | Percent of Fetuses/Dam Being Resorbed or Dead ± S.E.M. and (Number Early/Number Late)a | Percent of Fetuses/ Dam Having Cleft Palate ± S.E.M. and (Number Among Investigated) |
|---|---|---|---|---|
| DFMO + dioxane | 0 (0/7) | 9.3 ± 0.58 (65) | 9.2 ± 3.1 (6/0) | 0 (0/59) |
| TCAOB + saline | 100 (14/14) | 7.6 ± 0.68 (106) | 11.3 ± 4.3 (7/5) | 77.8 ± 8.2 (76/99) |
| TCAOB + DFMO | 75 (12/16) | 7.5 ± 0.60 (120) | 13.3 ± 3.4 (16.0) | 42.3 ± 7.5* (44/104) |
aResorbed plus dead fetuses (<6 mm of length) have been assigned to the group of early dead, while dead fetuses more than 6 mm of length have been assigned to the group of late dead.
* Significantly different from control (saline-plus TCAOB-treated), by Students's t-test, P < 0.005.
__________
Reference: Hassoun and Arif, 1988
- in ovo, chick(TCAOB)
-
°
Fertile eggs from a crossbred F1(NH X SCWL) were injected with
TCAB or TCAOB in corn oil at doses ranging from 0.1 ng to 100
µg/egg on day 4 or days 11-13 of incubation (volume of 20
µl per egg). Control eggs were injected with corn oil only.
Surviving embryos were incubated until hatching occurred.
An insignificant decrease in hatchability was observed in eggs injected with TCAB and TCAOB on day 4 compared to controls. The majority of deaths occurred before day 13 of incubation. Few deaths were observed in eggs injected on days 11-13. A 100% mortality was observed in eggs treated with 1.0 µg TCAB and 0.1 µg TCAOB on day 4 of incubation. An LD50 of 44 ng and 12 ng was estimated for TCAB and TCAOB, respectively.
Numerous malformations were detected in both hatched chicks and embryos that died prior to hatching. A direct causal relationship between TCAB and TCAOB exposure and rump edema was observed. Edema was detected in embryos and hatched chicks from eggs treated with 0.01 µg (3.4%) - 1.0 µg (4.2%) of TCAB and 0.005 µg (2.9%) - 0.05 µg (7.3%) of TCAOB. The highest incidence of rump edema in treated embryos occurred in the 0.0075 µg TCAOB/egg (22.5%) and 0.05 µg TCAB/egg (5.1%) treated fetuses. The percent of embryos with rump edema ranged from 2.5-5.1% (TCAB) and 2.1-22.5% (TCAOB). Rump edema was not observed in the control group. All embryos (except 2) with rump edema died before hatching. The authors concluded that TCAB and TCAOB are "extremely toxic and potentially teratogenic" in the chick. However, these compounds appear to be less toxic than TCDD [Schrankel et al., 1982].
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